In electrophoretic processes for separation of DNA components, DNA samples are placed in one or more wells located along one side edge of a thin rectangular gel plate. A voltage is applied to the gel, causing the DNA molecules to migrate out of the wells and travel laterally of the plate in the direction of applied voltage. Relative migration rates are determined largely by DNA component molecular weights, with molecules of lesser weight migrating more rapidly than molecules of greater weight. When completed after a predetermined time duration, the DNA samples are separated in a ladder series of bands extending laterally from the plate edge along which the sample wells are located. Each band contains DNA components of a specific molecular weight. Analysis therefore requires precise measurement of migration distant of, and molecular concentration within, the various bands.
Conventional techniques for analysis of electrophoretic records of the type described above typically contemplate use of dyes to facilitate optical analysis. For example, ethidium bromide is a fluorescent dye that is conventionally employed to facilitate analysis of DNA component concentration as a function of fluorescence of the individual bands under ultraviolet light. However, conventional analysis techniques, typically involving photographic recording of the fluorescing gel record, require extended exposure of the gel record, resulting in a record degradation. Specifically, conventional photographic procedures involve placement of the gel record on a transilluminating ultraviolet light table. Fluorescence is photographed using a suspended filter-equipped camera. While on the transilluminator, the gel is completely irradiated with ultraviolet light. Heat generated by the light causes the DNA to spread within the gel, resulting in loss of definition. Furthermore, the ultraviolet light itself causes damage to the DNA within the gel. This is a particularly serious problem when the DNA is to be recovered for other uses.
The photographic record is a second-generation record, which adds a source of error, not to mention time and expense, to the analysis process. Photographic measurements produce non-linear photometric results. This makes measuring DNA concentration very difficult. Furthermore, photographic film records require manual analysis, a process that is necessarily highly dependent upon training and skill of the technician. Thus, prior art techniques are at best amenable to qualitative analysis of electrophoretic DNA separation records, but are not readily amenable to precise quantitative analysis.
Another problem that inheres in prior art electrophoretic record analysis techniques lies in calibration of individual records and comparative analysis among a plurality of records. Because the analysis techniques are highly dependent upon skills of individual technicians, such records do not readily lend themselves to direct qualitative or quantitative comparison. Furthermore, the prior art has yet to propose an economical and reliable technique for quantitatively digitizing electrophoretic records for manipulation, analysis, and long term storage and retrieval in a digital computer.
It is therefore a general object of the present invention to provide a method and apparatus for analyzing electrophoretic DNA separations of the subject character that are economical to implement, that may be readily employed in both clinical and research applications by relatively unskilled personnel, and that facilitate quantitative analysis of DNA separations directly from the stained DNA in the gel separations themselves without intermediate photographic recording steps and the like.
Another and more specific object of the present invention is to provide a method and apparatus of the described character for scanning and converting electrophoretic records into digital format in which the records may be readily electronically manipulated and analyzed. Yet another object of the invention is to provide a method and apparatus of the described character that include facility for compensating the digital record against effects of background radiation and variations in scanning illumination intensity, such that normalized records may be employed directly for purposes of comparative analysis.
A further object of the invention is to provide apparatus for scanning and recording electrophoretic gel separation records of the subject type that includes facility for controlling scanning resolution, is readily adaptable for use in conjunction with electrophoretic gel separations of a variety of types, and that features a compact and modular design.